In age-related macular degeneration (AMD), retinal pigment epithelial (RPE) cells and photoreceptor cells of the retina degenerate, causing blindness. Individuals with AMD have elevated levels of iron in their retinas, which may contribute to AMD pathology by causing oxidative stress. To understand how iron accumulates in the retinas of individuals with AMD, we must understand the roles and functions of iron- handling proteins, such as Hephaestin (Hp), in the retina. Hp is found intracellularly in RPE cells, but its exact location and role are unknown. The first aim of this project is to determine the intracellular location and intracellular role of Hp in RPE cells. Confocal microscopy and immuno-EM will be used to localize Hp. Iron transport studies in mice with secondary ion mass spectroscopy (SIMS) will be employed to study Hp's role. Hp may play a role in iron transport from cell to cell. Intercellular iron transport in the retina is essential because not all retinal cells can obtain iron directly from blood vessels due to the blood-retina barrier. The second aim of this project is to study the role of Hp in intercellular iron transport. Light microscope autoradiography will be used. Systemic mutation of Hp and its homologue, Ceruloplasmin in Cp/sla mice, causes retinal iron accumulation and retinal degeneration in mice. Although these mice model some features of AMD such as accumulation of iron in RPE and photoreceptor cells, RPE hypertrophy, RPE and photoreceptor degeneration, choroidal neovascularization and immune cell infiltration, information from them is limited because they are not fertile and die early. The third aim of this project is to test if conditional knockout of Hp in RPE cells alone or photoreceptor cells alone can cause retinal iron accumulation and retinal degeneration. If so, these mice will be a superior retinal degeneration model as they will have full viability and fertility. Immunohistochemistry, electron micrcoscopy, atomic absorption spectroscopy, Western analysis, qPCR and Perls stain will be used to characterize the retinas of these mice. PUBLIC HEALTH RELEVANCE: AMD is the leading cause of blindness in individuals over age 55 in the United States. This work will help us understand the role of iron in AMD pathogenesis and will characterize a new animal model with features of AMD. This information and these animal models will be valuable for understanding AMD pathogenesis and testing therapies for AMD.